Simona Donati

IFN‐α‐conditioned dendritic cells are highly efficient in inducing cross‐priming CD8+ T cells against exogenous viral antigens

Dendritic cells (DC) generated after a short‐term exposure of monocytes to IFN‐α and GM‐CSF (IFN‐DC) are highly effective in inducing cross‐priming of CD8+ T cells against viral antigens. We have investigated the mechanisms responsible for the special attitude of these DC and compared their activity with that of reference DC. Antigen uptake and endosomal processing capabilities were similar for IFN‐DC and IL‐4‐derived DC. Both DC types efficiently cross‐presented soluble HCV NS3 protein to the specific CD8+ T cell clone, even though IFN‐DC were superior in cross‐presenting low amounts of viral antigens. Moreover, when DC were pulsed with inactivated HIV‐1 and injected into hu‐PBL‐SCID mice, the generation of virus‐specific CD8+ T cells was markedly higher in animals immunized with IFN‐DC than in mice immunized with CD40L‐matured IL‐4‐DC. Of interest, in experiments with purified CD8+ T cells, IFN‐DC were superior with respect to CD40L‐matured IL‐4‐DC in inducing in vitro cross‐priming of HIV‐specific CD8+ T cells. This property correlated with enhanced potential to express the specific subunits of the IL‐23 and IL‐27 cytokines. These results suggest that IFN‐DC are directly licensed for an efficient CD8+ T cell priming by mechanisms likely involving enhanced antigen presentation and special attitude to produce IL‐12 family cytokines.

IFN-α enhances cross-presentation in human dendritic cells by modulating antigen survival, endocytic routing, and processing.

Cross-presentation allows antigen-presenting cells to present exogenous antigens to CD8(+) T cells, playing an essential role in controlling infections and tumor development. IFN-α induces the rapid differentiation of human mono-cytes into dendritic cells, known as IFN-DCs, highly efficient in mediating cross-presentation, as well as the cross-priming of CD8(+) T cells. Here, we have investigated the mechanisms underlying the cross-presentation ability of IFN-DCs by studying the intracellular sorting of soluble ovalbumin and nonstructural-3 protein of hepatitis C virus. Our results demonstrate that, independently from the route and mechanism of antigen entry, IFN-DCs are extraordinarily competent in preserving internalized proteins from early degradation and in routing antigens toward the MHC class-I processing pathway, allowing long-lasting, cross-priming capacity. In IFN-DCs, both early and recycling endosomes function as key compartments for the storage of both antigens and MHC-class I molecules and for proteasome- and transporter-associated with Ag processing-dependent auxiliary cross-presentation pathways. Because IFN-DCs closely resemble human DCs naturally occurring in vivo in response to infections and other danger signals, these findings may have important implications for the design of vaccination strategies in neoplastic or chronic infectious diseases.

Interferon-α-Conditioned Human Monocytes Combine a Th1-Orienting Attitude with the Induction of Autologous Th17 Responses: Role of IL-23 and IL-12

IFN-α exerts multiple effects leading to immune protection against pathogens and cancer as well to autoimmune reactions by acting on monocytes and dendritic cells. We analyzed the versatility of human monocytes conditioned by IFN-α towards dendritic cell differentiation (IFN-DC) in shaping the autologous T-helper response. Priming of naïve CD4 T cells with autologous IFN-DC in the presence of either SEA or anti-CD3, resulted, in addition to a prominent expansion of CXCR3+ IFN-γ-producing CD4 Th1 cells, in the emergence of two distinct subsets of IL-17-producing CD4 T cells: i) a predominant Th17 population selectively producing IL-17 and expressing CCR6; ii) a minor Th1/Th17 population, producing both IL-17 and IFN-γ. After phagocytosis of apoptotic cells, IFN-DC induced Th17 cell expansion and IL-17 release. Notably, the use of neutralizing antibodies revealed that IL-23 was an essential cytokine in mediating Th17 cell development by IFN-DC. The demonstration of the IFN-DC-induced expansion of both Th1 and Th17 cell populations reveals the intrinsic plasticity of these DC in orienting the immune response and provides a mechanistic link between IFN-α and the onset of autoimmune phenomena, which have been correlated with both IL-17 production and exposure to IFN-α.

Anti-tumor CD8+ T cell immunity elicited by HIV-1-based virus-like particles incorporating HPV-16 E7 protein.

Here we report a novel strategy for the induction of CD8(+) T cell adaptive immune response against viral and tumor antigens. This approach relies on high levels of incorporation in HIV-1 VLPs of a mutant of HIV-1 Nef (Nef(mut)) which can act as anchoring element for foreign proteins. By in vitro assay, we found that VLP-associated Nef(mut) is efficiently cross-presented by antigen presenting cells. Inoculation in mice of VLPs incorporating the HPV-16 E7 protein fused to Nef(mut) led to an anti-E7 CD8(+) T cell response much stronger than that elicited by E7 recombinant protein inoculated with incomplete Freunds adjuvant and correlating with well-detectable anti-E7 CTL activity. Most relevantly, mice immunized with Nef(mut)-E7 VLPs developed a protective immune response against tumors induced by E7 expressing tumor cells. These results make Nef(mut) VLPs a promising candidate for new vaccine strategies focused on the induction of CD8(+) T cell immunity.

β-Glucan of Candida albicans cell wall causes the subversion of human monocyte differentiation into dendritic cells

The functional consequences of treating human monocytes with purified and chemically characterized Candida albicans β‐glucan—a major microbial pathogen associated molecular pattern—on their differentiation into dendritic cells (DC) were investigated. We show here that β‐glucan‐treated monocytes differentiated into mature DC (Glu‐MoDC) with altered phenotype and functional behavior, similarly to DC derived from C. albicans germ‐tubes‐infected monocytes (Gt‐MoDC). They failed to express CD1a and to up‐regulate CD80 and DR molecules. Moreover, they produced IL‐10 but not IL‐12 and primed naive T cells without inducing their functional polarization into effector cells. Since C. albicans β‐glucan is a mixture of both β‐(1,3) and β‐(1,6) glucan, we investigated their relative contribution by the use of non‐Candida β‐glucan structural analogs. We found that high molecular weight (MW) glucans β−(1,6) pustulan and β‐(1,3) curdlan totally mimicked the effect of C. albicans β‐glucan, while the low MW β‐(1,3) glucan laminarin did not have any effect. Because β‐glucan is a common constituent of all fungi and is abundantly released in vivo during systemic fungal infection, this novel effect of β‐glucan has potential implications for host‐parasite relationship in candidiasis and other mycoses. In particular, our data suggest that β‐glucan could bias noninfected, bystander monocytes, thus aggravating the general immunodeficiency, predisposing to systemic fungal infection.

Negatively charged gold nanoparticles as a dexamethasone carrier: stability in biological media and bioactivity assessment in vitro

Gold nanoparticles (AuNPs) have been extensively used in biological applications because of their high biocompatibility, ease of characterization and the extensive knowledge of their surface chemistry. These features make AuNPs readily exploitable for drug delivery and novel diagnostic and therapeutic approaches. In a previous work, we showed that small size (5–10 nm) AuNPs functionalized by sodium 3-mercapto-1-propanesulfonate (3MPS) can be efficiently loaded with the glucocorticoid drug dexamethasone (DXM) and are stable in water and PBS. In the present study, we further analysed the stability and the drug release kinetics of DXM-loaded AuNPs functionalized by sodium 3-mercaptopropane sulfonate (AuNP-3MPS/DXM) and their unconjugated counterparts (AuNP-3MPS) in different biological media. Moreover, we evaluated AuNP-3MPS cyto-compatibility on two mammalian cell lines and tested their specific activity as drug carriers on DXM-sensitive murine and human tumor cells. The colloidal stability of AuNP-3MPS/DXM was significantly increased in all tested culture media, compared with the unconjugated AuNP-3MPS and both AuNP-3MPS formulations which proved non-toxic to biological systems in vitro. Most importantly, we showed that AuNP-3MPS/DXM continuously release bioactive DXM molecules that efficiently induce cell proliferation arrest and apoptotic cell death on a human lymphoma cell line and upregulation of the DXM-inducible programmed cell death-1 (PD-1) molecule on activated mouse T lymphocytes. These data confirm that the AuNP-3MPS/DXM conjugate is a promising system for drug delivery and open interesting perspectives for future in vivo applications.

NK Cell Activation in the Antitumor Response Induced by IFN-α Dendritic Cells Loaded with Apoptotic Cells from Follicular Lymphoma Patients

Follicular lymphoma (FL) is the most common form of indolent non-Hodgkin lymphoma. This malignancy is considered virtually incurable, with high response rates to therapy but frequent relapses. We investigated the ability of monocyte-derived dendritic cells generated in the presence of IFN-α and GM-CSF (IFN-DC) and loaded with apoptotic lymphoma cells to activate immune responses against FL cells, with the ultimate goal of designing novel patient-specific vaccination strategies for the treatment of FL. In this article, we show that apoptotic tumor cell–loaded IFN-DC from FL patients, which were cultured for 2 wk with autologous lymphocytes, led to Th1 response skewing, based on significantly higher levels of IFN-γ production and a remarkable increase in CD8+ and NK cell frequency, consistent with the detection of enhanced cytotoxic effector function toward autologous FL cells. IFN-DC were found to promote efficient NK cell activation, increased expression of cytotoxicity receptors, and extensive IFN-γ production in the virtual absence of IL-10. Moreover, direct recognition and killing of primary autologous lymphoma cells by activated NK cells from FL patients was also demonstrated. A critical role was demonstrated for MHC class I–related chain A and B and membrane-bound IL-15 in IFN-DC–mediated NK cell activation and early IFN-γ production. The overall results indicate that IFN-DC loaded with autologous apoptotic FL cells represent a valuable tool for improving the potency of therapeutic cancer vaccines through the efficient induction of NK cell activation and promotion of CD8+ T cell antitumor immunity.